山羊骨骼肌细胞分化中H19-MyoD轴相关的核心lncRNAs调控网络构建

Muscle cell differentiation is a dynamic process precisely controlled by the interactions among multiple factors. As the most important regulatory RNA, the functional research of lncRNAs is still in the emerging stage, and nowadays the popular study method focuses on one direction (from lncRNAs gene to protein gene, or vice versa), which result in a certain gap from reciprocal regulation characteristics between genes in reality. Taking advantage of the mutual regulationary interactions between MyoD (protein gene) and H19 (lncRNA gene), the core factors regulating gene expression genome-widely during muscle differentiation, and skeletal muscle satellite cells of meaty goat as material, and methods including RNA-seq, CHIP and FISH etc., we will investigate 1）characteristics and expression profiles of goat H19 gene; 2）characterize H19 – MyoD-axis related lncRNAs in regulating skeletal muscle differentiation, and analyze five to eight key lncRNAs well; and 3）construct an H19 - MyoD –axis core regulatory network of skeletal muscle differentiation which harbors several lncRNAs, miRNAs and encoding genes. This study will vastly elevate lncRNAs research level from the point-line model to the surface network mode in muscle differentiation. The H19 – MyoD-axis regulation network constructed will reflect the comprehensive relationships between genes in cell more precisely and found wonderful insights into the muscle growth regulatory mechanisms.

肌细胞分化是由多种因子互作调控的动态过程，其中最重要的调控因子lncRNA功能解析尚处于新兴阶段且通用的研究方法是单向的（lncRNA到蛋白基因或反过来），与细胞内相互调控的机制存在差距。本研究利用肌细胞分化发育中具有全基因组调控能力并相互调控的核心因子H19(lncRNA)和MyoD（蛋白基因），以山羊骨骼肌卫星细胞为材料，整合转录组测序、CHIP-seq、FISH等技术，1）揭示山羊H19基因序列特点和表达规律；2）解析H19-MyoD轴调控骨骼肌分化的lncRNAs基因数目和特点，深度分析其中~5个核心lncRNAs基因；3）构建以H19-MyoD为轴，由lncRNAs、miRNAs以及编码基因组成的细胞分化调控网络。本研究将lncRNAs功能分析由目前的点线模式提升到网络模式；所构建H19-MyoD调控网络将能更准确反应细胞内基因间关系，是全面深入了解肌肉生长发育的重要基础。

骨骼肌细胞的生成调控是一个集合转录因子和调控性非编码RNA的动态网络。虽然目前对蛋白编码基因和miRNA的研究较丰富，但lncRNAs和circRNA等尚处于新兴阶段。本研究利用肌细胞分化发育中具有关键作用的核心因子 H19(lncRNA)和MyoD(蛋白基因)，以山羊骨骼肌卫星细胞为材料，整合转录组测序、RIP、FISH等技术，获得了骨骼肌分化中受到H19调控的mRNA和多种非编码RNA，揭示了参与成肌分化的分子信号途径，构建了多维的调控通路。主要结果如下：.（1）利用RACE技术分析发现山羊H19基因生成2个转录本，主要在肌肉组织中表达。H19基因序列在物种间保守性较差，且山羊、绵羊和牛的H19不具有产生miR-675的能力。MyoD蛋白可结合在H19基因启动子区和外显子区域激活H19表达，而H19促进山羊骨骼肌卫星细胞成肌分化。.（2）获得大量在肌细胞分化中受到H19调控的circRNAs、miRNA、mRNA和lncRNA，主要富集到TGF-β信号通路(TGF-beta signaling pathway)、cAMP信号通路(cAMP signaling pathway)、Ras信号通路(Ras signaling pathway)以及MAPK信号通路(MAPK signaling pathway)，在骨骼肌发育过程中起重要作用。.（3）完成了H19相关的lncRNA/circRNA-miRNA-mRNA调控网络构建，发现miR-125a/b、miR-133和miR-7等介导了多个调控网络。筛选并验证了lncR-125b/miR-125b/IGF2、linc-MD1/miR-133a/SRF、lnc_594/133a-3p/FGFR1以及miR-193b-3p/IGF2BP1等通路对肌细胞分化的作用。.（4）发现H19与MyoD mRNA在肌细胞的增殖或分化阶段均共定位，并且利用MyoD RNA序列可有效地富集H19或者其片段，初步证明H19可结合MyoD mRNA调控成肌。.综上所述，本项目对H19-MyoD相关调控网络的分析与验证，将能更准确反应细胞内基因间关系，是全面深入了解肌肉生长发育的重要基础。本研究结果将对进一步研究哺乳动物肌肉的生长发育或肌病等具有重要的参考价值。